In planta modification of the potato tuber cell wall

Apart from its well known uses in the human diet a large amount of the grown potatoes (about one third in the Netherlands) is used for the isolation of starch which is used in several food and non-food applications. The cell wall fibres comprise a large portion of the waste material remaining after the starch isolation process. While cell wall fibres from some other plant species are used in food and non-food industry, a structural alteration of the potato fibres is necessary before similar applications are possible. However, before it is possible to generate plants with a tailor-made cell wall composition, questions concerning the best approach have to be answered and the necessary tools will have to be identified and made available. This thesis describes the results of a study investigating the possibilities to generate transgenically modified potato plants with an altered cell wall composition. These experiments were mostly focussed on altering pectin composition. This is particularly interesting because several studies already showed that many different pectin structures occur in specific plants, plant tissues and developmental stages. Plants with a specific alteration in pectin structure may aid in revealing the biological significance of these different structures. Additionally, the possibility to produce a particular pectin structure may be useful for the food industry, in which pectins from other plant species are already used as a gelling agent.At the start of the work described in this thesis only few genes involved in cell wall biosynthesis were identified which favoured the heterologous expression of fungal pectin degrading enzymes. A rhamnogalacturonan lyase ( e RGL) from Aspergillus aculeatus , which is able to cleave the rhamnogalacturonan I (RG I) at specific sites, was introduced. The e RGL was successfully expressed (under control of the granule-bound starch synthase promoter) and translated into an active protein, demonstrated by e RGL activity in the tuber extracts. These tubers showed clear morphological alterations, including radial swelling of the periderm cells and development of intercellular spaces in the cortex. Sugar compositional analysis and antibody labelling studies showed a large reduction in galactan and arabinan side-chains of RG I. These data show the possibility of specifically modifying cell wall polysaccharide structures by the introduction of such a pectin degrading enzyme. Additionally, the results suggest that RG I has an important role in anchoring galactans and arabinans at particular regions in the wall and in normal development of the periderm. The utility of these transgenic plants in answering questions concerning the biological importance of cell wall polysaccharides is evident.Apart from modifying the cell wall composition by the introduction of pectin degrading enzymes two experiments were performed focussed on interference with the biosynthetic machinery of the plant cell wall at different levels. The first study concerns the modulation of cellulose synthase ( CesA ) gene expression. Since this enzyme is polymerising theb-1,4 glucan chains forming cellulose, its altered expression is likely to directly affect the level of cellulose in the wall. In the second study the expression of the UDP-Glc-4-epimerase ( UGE ) was modulated. The UGE is responsible for the conversion of UDP-glucose to UDP-galactose and vice-versa. Its altered expression is likely to affect the amount of cell wall bound galactan.Four CesA genes were isolated from potato and one full length cDNA clone was used for up- and down-regulation of the corresponding RNA expression levels controlled by the granule-bound starch synthase promoter. Fourier Transform Infra-Red microspectroscopy (FTIR) was used for the identification of transformants with altered levels of cellulose in their tuber cell walls in comparison to WT plants. A further quantification of these results, by measuring the cellulose content in the cell wall material, showed that by modulating the CesA expression levels, tubers with levels of cellulose ranging from 50 to 200% of the WT amount were obtained. Especially the increase in cellulose is quite remarkable and in contradiction with the general believe that expression of more than one CesA gene (and possibly even more genes) is necessary to achieve such a modification. By using a specific region of the other three CesA genes in antisense experiments we managed to individually down regulate these genes and concomitantly the cellulose levels in the tubers of these plants. The use of this so-called class specific region (CSR), which is only present in plant cellulose synthases and is believed to determine the genetic difference between the different CesA genes in one plant, showed to be sufficient to down regulate the corresponding gene. In contrast to many other plants and plant systems, depletion of cellulose (to 50% of WT level) in potato tubers did not result in any phenotypic alterations. However, our potato plants were grown at normal conditions while some of the cellulose synthase mutants only revealed a phenotype when grown at restrictive conditions. Another important result is the fact that not all potato transformants with decreased cellulose levels show modifications in their pectin composition. This indicates a delicate balance between cellulose and pectin levels and that an altered pectin composition in plants with depleted cellulose is not necessarily a response upon reduced strength of the cell wall.For the UDP-Glc-4-epimerase two potato cDNA clones ( StUGE45 and StUGE51 ) were identified and used for overexpression in potato tubers. The increased levels of cell wall bound galactan in these tubers indicates the importance of UDP-galactose levels for galactan deposition in the cell wall. Additionally these plants showed a small decrease in the amount of galacturonan. This suggests that alterations in the UDP-galactose pool size can influence the levels of nucleotide sugars which are used for the synthesis of other polysaccharides. Additionally, the elevated expression levels of the two UGE s showed to have different effects, which suggests that they have a different function in plant development. Further research has to show whether other polysaccharides than cell wall galactan are affected by this decrease in galactose. Xyloglucan and galactomannan also contain galactosyl residues and the decrease does not necessarily affect the levels of RG I bound galactan.Both these studies show the possibility to induce alterations in the cell wall composition by interfering with the biosynthetic machinery. Identification of more genes involved in cell wall biosynthesis is necessary to enable new studies in the future. An RNA fingerprinting experiment was performed to investigate the possibility of identifying new genes involved in primary cell wall biosynthesis. Potato leaf protoplasts showed to regenerate a new cell wall in the first 18h after transfer to a culture medium. At 5 distinct time-points RNA was isolated and the expressed genes were visualised using cDNA-AFLP. Around 8500 transcript derived fragments (TDFs) were visualised from which 156 were isolated and sequenced. However, no cell wall related TDFs were identified. This indicates that even though the protoplasts actively regenerate a new cell wall, this did not result in highly increased expression of genes involved in cell wall biosynthesis or modification.In summary the experiments described in this thesis showed that different approaches can be used to generate a modified cell wall composition in potato tubers. These genetically modified plants have shown to be an interesting study material for unravelling the biological function of different cell wall polysaccharide structures. Additionally these transformants obviously showed that the potato tuber cell wall is amenable to genetic modification. There is a wide range of wall modifications which is tolerated by the tubers, which may hold a promise for the future in valorising the fibre fraction of potato after starch isolation.</p

Exploring the use of cDNA-AFLP with leaf protoplasts as a tool to study primary cell wall biosynthesis in potato

An RNA fingerprinting study of potato leaf protoplasts was performed to explore its suitability for identifying candidate genes involved in primary cell wall biosynthesis. Microscopic analysis, using calcofluor white to stain cellulose, showed that the protoplasts generated a new cell wall in the first 18 h after transfer to a culture medium. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) was used to visualise differential gene expression at five distinct time-points within these first 18 h. In vitro plants (with and without exposure to severe physical damage) served as controls. Around 8500 transcript derived fragments (TDFs) were visualised which showed varying expression patterns in the protoplasts and controls. In total 156 TDFs were isolated, sequenced and used to search for homologies. Over 50% of these TDFs showed homology to described genes, involved in several general plant processes. However, only one cell wall related TDF (a pectin esterase) was found. Our results showed that even though the protoplasts actively regenerate a new cell wall, this did not result in highly increased expression of genes involved in cell wall biosynthesis or modification. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved

Pectin : the hairy thing : evidence that homogalacturonan is a side chain of rhamnogalacturonan I

In vitro degradation studies of pectic polysaccharides with novel fungal pectinases, investigations in which these polymers were treated with dilute acid, and microscopic analysis of extracted pectins have provided clues on how these polysaccharides are linked. Therefore it is believed that pectin is not an extended backbone consisting of homogalacturonan and rhamnogalacturonan regions, but rather a rhamnogalacturonan with neutral sugar and homogalacturonan side chain

Antisense suppression of a potato alpha-SNAP homologue leads to alterations in cellular development and assimilate distribution

Using the cDNA-AFLP method, we have isolated a transcript-derived fragment (TDF) which shows a differential expression pattern during tuber organogenesis of Solanum tuberosum L. The TDF was used to isolate a cDNA clone carrying a 1.5 kb insert and potentially coding for a 32.5 kDa peptide which, by homology, represents a potato homologue of an alpha-snap gene and has been designated Stsnap. Northern analysis showed that the Stsnap gene is expressed in actively dividing tissues throughout the potato plant. Analysis of genomic DNA from potato revealed that the Stsnap gene is likely to be a single-copy gene. The expression of antisense Stsnap cDNA under the control of the CaMV 35S promoter results in plants with an altered morphology such as curled leaves. Several of these transgenic lines also display cellular and developmental abnormalities with distinct changes in assimilate transport including accumulation of starch and soluble sugars in source leaves. We argue that these findings are consistent with the hypothetical function of the StSNAP gene product in vesicle targeting and fusion during plant development

Reduction of starch granule size by expression of an engineered tandem starch-binding domain in potato plants

Granule size is an important parameter when using starch in industrial applications. An artificial tandem repeat of a family 20 starch-binding domain (SBD2) was engineered by two copies of the SBD derived from Bacillus circulans cyclodextrin glycosyltransferase via the Pro-Thr-rich linker peptice from Xyn10A from Cellulomonas fimi. SBD2 and a single SBD were introduced into the amylose-free potato mutant, amf, using appropriate signal sequences. The accumulation of SBD2 into transgenic starch granules was much higher than that of SBD. In a number of transformants, particularly amfSS3, the starch granules were much smaller than in control plants. The amfSS3 mean granule size was 7.8 mum, compared with 15.2 mum in the control, whereas other starch properties were unaltered. This new starch combines the advantage of the high purity of potato starch with that of the small granule size of other crop species, such as cassava, taro and wheat. This starch may find application in the manufacture of biodegradable plastic films. Both genes were also expressed in Escherichia coli and the affinity for soluble starch of the purified recombinant proteins was determined. SBD2 had an approximately 10-fold higher affinity for starch than SBD, indicating that the two appended SBDs act in synergy when binding to their target polysaccharide ligand

Overexpression of two different potato UDP-Glc-4-epimerases can increase the galactose content of potato tuber cell walls

UDP-glucose 4-epimerases (UGE) catalyze the reversible conversion of UDP-glucose to UDP-galactose. Two potato UGE genes (StUGE45 and StUGE51) were isolated from a potato cDNA library using the Arabidopsis thaliana UGE1 cDNA as a probe. The cDNA clones cluster differently in a phylogenetic tree of plant epimerases. StUGE51 showed higher expression than StUGE45, particularly, in older tubers, flowers, stems and in vitro plants. Transgenic potato plants were generated to examine the effect of modified UGE expression on the amount of cell wall galactose. Sense expression of the two StUGE clones in potato gave plants with no overt developmental phenotype. In contrast to WT plants, a large number of transformants had the ability to overcome the toxic effect of galactose in the culture medium during in vitro growth. The UGE45 transformants showed a clear correlation of increased UGE RNA expression levels with this tolerance to galactose, whereas the UGE51 transformants did not. The elevated UGE expression in the transformants resulted in an increased galactose content in potato tuber cell walls. This effect was more profound for the UGE45 transformants than for the UGE51 ones. Our results suggest that the two potato UGEs have different biochemical properties, and that they have a different function in plant development. (C) 2004 Elsevier Ireland Ltd. All rights reserved

Functional characterization of NRAMP3 and NRAMP4 from the metal hyperaccumulator Thlaspi caerulescens

The ability of metal hyperaccumulating plants to tolerate and accumulate heavy metals results from adaptations of metal homeostasis. NRAMP metal transporters were found to be highly expressed in some hyperaccumulating plant species. Here, we identified TcNRAMP3 and TcNRAMP4, the closest homologues to AtNRAMP3 and AtNRAMP4 in Thlaspi caerulescens and characterized them by expression analysis, confocal imaging and heterologous expression in yeast and Arabidopsis thaliana. TcNRAMP3 and TcNRAMP4 are expressed at higher levels than their A. thaliana homologues. When expressed in yeast TcNRAMP3 and TcNRAMP4 transport the same metals as their respective A. thaliana orthologues: iron (Fe), manganese (Mn) and cadmium (Cd) but not zinc (Zn) for NRAMP3; Fe, Mn, Cd and Zn for NRAMP4. They also localize at the vacuolar membrane in A. thaliana protoplasts. Inactivation of AtNRAMP3 and AtNRAMP4 in A. thaliana results in strong Cd and Zn hypersensitivity, which is fully rescued by TcNRAMP3 or TcNRAMP4 expression. However, metal tolerance conferred by TcNRAMP expression in nramp3nramp4 mutant does not exceed that of wild-type A. thaliana. Our data indicate that the difference between TcNRAMP3 and TcNRAMP4 and their A. thaliana orthologues does not lie in a different protein function, but probably resides in a different expression level or expression patter